Method for PR-39 peptide mediated selective inhibition of IkappaBalpha

ABSTRACT

The present invention provides both a method and means for regulating IκBα degradation, NFκB activity, and NFκB-dependent gene expression within living cells, tissues, and organs in-situ. The selective regulation is performed using native PR-39 peptide or one of its shorter-length homologs, for interaction with such IκBα and proteasomes as are present in the cytoplasm of viable cells. The result of PR-39 peptide interaction with IκBα is a selective alteration in the intracellular proteolytic activity of proteasomes, which in turn, causes a reduction of IκBα, a decrease of NFκB activity, and a down-regulation of NFκB-dependent gene expression.

CROSS REFERENCE

[0001] The present application is a Continuation-In-Part of priorpending U.S. patent applications Ser. Nos. 09/276,868 filed Mar. 26,1999 and 09/426,011 filed Oct. 25, 1999.

RESEARCH SUPPORT

[0002] The research effort for the invention was supported in part bythe National Institutes of Health, grants HL 53793 and HL 56993 (MS), DK31396 (MS), GM51923 and GM 46147 (ALG), F32HL 10013 (RV), and in part bya grant from Chiron Corporation. The government has certain rights inthe invention.

FIELD OF THE INVENTION

[0003] The present invention is concerned generally with the Rel/NFκBfamily of transcription factors within viable cells comprising livingtissues and organs; and is particularly directed to proteasomemechanisms regulated by PR-39 peptides which result in a selectiveinhibition of IκBα degradation on-demand and may be used for controlledsuppression of NFκB transcription factor activity and NFκB-dependentgene expression.

BACKGROUND OF THE INVENTION

[0004] Degradation of proteins in mammalian cells proceeds via twodistinct pathways, the lysosome-dependent and proteasome-dependentsystems. The proteasome-dependent system catalyzes the hydrolysis ofproteins marked for degradation typically by conjugation to ubiquitin,but also can degrade certain non-ubiquitinated proteins as well.

[0005] Proteasome-mediated degradation is also a principal means forcontrolling the intracellular levels of most cell proteins, includingthe recognized major regulators of gene expression such as NFκBtranscription factor; the inhibitor protein IκBα; hypoxia-inducingfactor (HIF)-1a; protooncogenes c-Fos, c-Jun and c-Mos; and the variouscyclins. See for example: Whiteside, S. T. and A. Israel, Semin. CancerBiol. 8: 75-82 (1997); Srinivas et al., J. Biol. Chem. 273: 18019-18022(1998); Salceda, S. and J. Caro, J. Biol. Chem. 272: 22642-22647 (1997);Huang et al., Proc. Natl. Acad. Sci. USA 95: 7987-7992 (1998); He etal., J. Biol. Chem. 273: 25015-25019 (1998); and Pahl, H. L. and P. A.Baeverle, Curr. Opin. Cell Biol. 8: 340-347 (1996). Furthermore, thesmaller-sized peptides generated by the proteasome during the course ofprotein breakdown are often biologically active; for example, somepeptides are presented as antigens on the class I majorhistocompatibility complex (MHC) [Rock, K. L. and A. L. Goldberg, Annu.Rev. Immunol. 17: 739-779 (1999)]. These degradation products thus cancause different effects and major consequences in a variety of cellularprocesses, many of which have substantive clinical value.

[0006] In particular, NFκB-dependent gene expression is recognized asplaying an important role in a number of biological processes of majormedical importance including immune, inflammatory and anti-apoptoticresponses [Baeuerle, P. A. and D. Baltimore, Cell 87: 13-20 (1996); Beg,A. A. and D. Baltimore, Science 274: 782-784 (1996); and Antwerp et al.,Science 274: 787-789 (1996)]. NFκB is a dimer molecule composed of thep50 and p65 (RelA) monomer subunits; and binding of this dimer complexto IκB inhibitor protein in a cytosol is believed to be the maincellular mechanism preventing NFκB-dependent transcription of genesunder normal conditions. A number of different extracellular stimuli(including TNFα, I1-1 and lipopolysaccharide) can trigger NFκBtranscription factor activation, most notably by causing a rapiddegradation of IκB inhibitor protein by the ubiquitin (Ub)-proteasomedegradation pathway.

[0007] Several steps necessary for proteasome-mediated IκBα degradationto occur have been identified. These include: phosphorylation of IκBα attwo sites by a specific IκBα kinase of the SCF1 family [Whiteside, S. T.and A. Ismael, Semin. Cancer Biol. 8: 75-82 (1997); Chen et al., Cell84: 853-862 (1996)]; the ubiquitination of the phosphorylated IκBα by aspecific E3 enzyme complex [Suzuki et al., Biochem. Biophys. Res. Comm.256: 121-126 (1996); Spencer et al, Genes Dev. 13: 284-294 (1999); Krollet al., J. Biol. Chem. 274: 7941-7945 (1999); Yaron et al., Nature 396:590-594 (1998); and Gonen et al., J. Biol. Chem. 274: 14923-14830(1999)]; and the subsequent binding to VCP (valosin-containing protein)that results in a physical link between the ubiquitinated IκBα and theproteasome.

[0008] Separate and distinct from these events is the PR-39 protein.PR-39 is a highly basic arginine/proline-rich peptide originallyisolated from porcine intestine on the basis of its anti-bacterialactivity [Agerbeth et al., Eur. J. Biochem. 202: 849-854 (1991)]. ThePR-39 peptide is secreted in a prepro-protein form that includes acanonical leader sequence and rapidly undergoes cleavage of theN-terminal portion to generate the mature form composed of the 39C-terminal amino acids [Gudmundsson et al., Proc. Natl. Acad. Sci. USA92: 7085-7095 (1995)]. While the sequence of the N-terminal part of theprepro-protein is highly homologous to the cathelin gene family members,the sequence of the 39 C-terminal amino acids that make up the maturepeptide, has no homology to any other known protein.

[0009] Research investigations have shown that PR-39 protein, canrapidly cross cell membrances; and, by virtue of its proline-richcomposition, may interact with SH3 domains of p47^(phox) and p130^(Cas)[Ross et al., Proc. Natl. Acad. Sci. USA 93: 6014-6018 (1996); and Chan,Y. R. and R. L. Gallo, J. Biol. Chem. 273: 28978-28985 (1998)]. ThePR-39 peptide (predominantly produced by blood-derived macrophages) isfound at the sites of active inflammation including skin wounds andmyocardial infarction and is seen as playing an important role byinducing expression of heparan sulfate-carrying core proteins, syndecan1 and 4 [Li et al., Circ. Res. 81: 785-796 (1997); and Gallo et al.,Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994)] and inhibitingdegradation of the hypoxia-inducible factor (HIF)-1α protein. However,the molecular events and mechanism of action involved in this peptide'sactions remain largely unknown.

[0010] Accordingly, although there have been many investigations,publications, and developments of these various entities, there remainsa general ignorance and failure of understanding by researchinvestigators and clinicians alike regarding useful and effectivespecific means and methods for suppressing NFκB-dependent geneexpression on-demand within living cells, tissues, and organs. Thus,while the value and desirability of selectively controlling NFκBtranscription factor activity—especially within cells at localizedtissue areas on an as-needed basis for individual subjects—is wellrecognized, these aims have remained a long-sought goal yet to beachieved to date in a practical manner.

SUMMARY OF THE INVENTION

[0011] The present invention has multiple aspects and uses. A firstaspect provides a method for selectively inhibiting degradation of IκBαwithin a targeted collection of viable cells in-situ, said methodcomprising the steps of:

[0012] identifying a collection of cells comprising viable cells in-situas a target for inhibiting IκBα degradation;

[0013] providing means for effecting an introduction of at least onemember selected from the group consisting of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells;

[0014] introducing at least one member of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells usingsaid effecting means;

[0015] allowing said introduced PR-39 oligopeptide collective member tointeract with such IκBα and proteasomes as are present within thecytoplasm of said targeted collection of cells whereby

[0016] (a) at least some of the proteasomes interact with said PR-39oligopeptide collective member,

[0017] (b) at least a part of the proteolytic activity mediated by saidinteracting proteasomes becomes selectively altered, and

[0018] (c) the selectively altered proteolytic activity of saidproteasomes results in a marked inhibition of IκBα degradation in-situwithin the cytoplasm of said targeted collection of viable cells.

[0019] A second aspect of the invention provides a method for decreasingthe activity of NFκB transcription factor in-situ within a collection ofviable cells, said method comprising the steps of:

[0020] identifying a collection of cells comprising viable cells in-situas a target for decreased NFκB activity;

[0021] providing means for effecting an introduction of at least onemember selected from the group consisting of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells;

[0022] introducing at least one member of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells usingsaid effecting means;

[0023] allowing said introduced PR-39 oligopeptide collective member tointeract with such IκBα and proteasomes as are present within thecytoplasm of said targeted collection of cells whereby

[0024] (a) at least some of the proteasomes interact with the PR-39oligopeptide collective member,

[0025] (b) at least a part of the proteolytic activity mediated by saidproteasomes becomes selectively altered by said interaction,

[0026] (c) the selectively altered proteolytic activity of saidproteasomes results in a marked reduction of IκBα degradation in-situwithin the cytoplasm of said targeted collection of cells; and

[0027] (d) said reduction of IκBα degradation results in a decrease inactivity for such NFκB transcription factor as is presentintracellularly.

BRIEF DESCRIPTION OF THE FIGURES

[0028] The present invention may be more fully understood and betterappreciated when taken in conjunction with the accompanying drawing, inwhich

[0029] FIGS. 1A-1F are presentations of empirical data showing theinteraction between PR-39 peptide and proteasomes and the effect of suchinteraction on IκBα degradation and NFκB activity;

[0030] FIGS. 2A-2D are presentations of empirical data showing theeffect of PR-39 peptide upon inhibition of NFκB-dependent geneexpression in cell culture and mice;

[0031] FIGS. 3A-3D are presentations of empirical data showing theselective nature of the PR-39 peptide effects; and

[0032] FIGS. 4A-4D are presentations of empirical data showing thatPR-39 peptide administration does not affect IκBα phosphorylation orubiquitination.

DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention is a method for selectively inhibiting thedegradation of IκBα protein via the purposeful introduction of nativePR-39 peptide or a member of the PR-39 derived oligopeptide family tothe cytoplasm of viable cells in-situ. The PR-39 peptide or the derivedmember of the oligopeptide family will interact with such IκBα proteinand proteasomes as are present intracellularly; and the consequence ofPR-39 peptide/IκBα/proteasome interactions is the marked inactivation ofNFκB transcription factor such that intracellular NFκB-dependent geneexpression is diminished and suppressed in-situ.

[0034] A number of major benefits and advantages are therefore providedby the means and methods comprising the present invention. These includethe following:

[0035] 1. The present invention provides means for selective inhibitionof IκBα protein degradation in-situ. By definition, therefore, bothin-vivo and in-vitro circumstances of use and application are envisionedand expected. Moreover, the viable cells which are the location of PR-39peptide, IκBα and proteasome interactions, alternatively may be isolatedcells; be part of living tissues comprising a variety of different cellssuch as endothelial cells, fibrocytes and muscle cells; and may alsocomprise part of specific organs in the body of a living human or animalsubject. While the user shall choose the specific conditions andcircumstances for practicing the present invention, the intended scopeof application and the envisioned utility of the means and methodsdescribed herein apply broadly to living cells, living tissues,functional organs and systems, as well as the complete living body unitas a viable whole.

[0036] 2. The present invention has a variety of different applicationsand uses. Of clinical and medical interest and value, the presentinvention provides the opportunity to selectively control NFκB-dependentgene expression in tissues and organs in a living subject which hassuffered defects or has undergone anoxia or infarction. A typicalclinical instance is the myocardial infarction or chronic myocardialischemia of heart tissue in various zones or areas of a living humansubject. The present invention thus provides opportunity and means forspecific site control of gene expression in NFκB-dependent cells. Thepresent invention also has major research value for researchinvestigators in furthering the quality and quantity of knowledgeregarding the mechanisms controlling NFκB-initiated gene expressionunder a variety of different conditions and circumstances.

[0037] 3. The present invention envisions and permits a diverse range ofmeans for introducing native PR-39 peptide or a shorter-length peptideof the oligopeptide family to a diverse range of different cells at aspecific location, site, tissue, organ, or system in the living body. Avariety of different routes of administration are available to thepractitioner which will vary with the type of cells and their location;and a wide and useful choice of delivery systems are conventionallyavailable, and in accordance with good medical practice, are adaptabledirectly for use. In this manner, not only are the means for PR-39peptide introduction under the control of the user, but also the mannerof application and the location of PR-39 peptide introduction can beprechosen and controlled.

I. Underlying Mechanisms for Initiating a Reduction of IκBα Degradation

[0038] The present invention utilizes and relies upon novel andpreviously unknown direct and indirect mechanisms of interaction betweenPR-39 peptide (or its shorter-length homologs) and proteasomes in-situas the basis for reducing IκBα protein degradation in cells, livingtissues, and organs. Evidence of such intracellular interactions isprovided by the experiments and empirical data described hereinafter.Such interactions between proteasomes, ubiquitinated IκBα and the PR-39family of peptides collectively (of any size) are previously unknown; infact, no meaningful relationship or interaction between any peptidewhatsoever and control of intracellular proteasome function has everbeen proposed or envisioned before the present invention was conceivedor demonstrated empirically.

[0039] As shown experimentally hereinafter, the PR-39 peptide (and theshorter-length PR-39 derived oligopeptide family members) whenintroduced into the cytoplasm of viable cells will interact, directlyand indirectly, with proteasomes. In some instances, the interactionbetween the collective of PR-39 oligopeptides and the proteasome isdirect. One example is a direct binding of PR-39 peptide with the α7subunit of the proteasome. In these instances, no intermediaries orcofactors are involved in the binding reaction; and such direct bindinginteractions result in a selective inactivation and inhibition ofproteasome function intracellularly such that expression of certainproteins such as HIF-1α is increased and stimulation of angiogenesissubsequently occurs.

[0040] In addition, however, alternative mechanisms of interaction formembers of the PR-39 peptide collective (including native PR-39 and itssubstituted or homologous forms) in addition to and other than directinactivation of a proteasome subunit concurrently exist and are often ineffect in-situ. As an examplary and representative instance illustratingan alternative mechanism of action, the experiments and empirical datapresented hereinafter demonstrate that: PR-39 peptide acts as aselective inhibitor of IκBα protein degradation by proteasomes; and thatPR-39 peptide selective inhibition of IκBα degradation is rapidlyreversible (unlike the action of known inhibitory compounds such aslactacysin); and that, even in the presence of PR-39 peptide, theintracellular expression of other proteasome-regulated proteins (such asp105 and p50) NFκB remain unchanged. These empirically documentedfindings are meaningful evidence of, harmonious with, and consistentdemonstrations of indirect mechanisms of interaction rather than anydirect action effect. Accordingly, the selective and reversible natureof PR-39 peptide activity with regard to inhibiting IκBα degradation isdeemed to be due to a prevention of recognition for the ubiquitinatedIκBα complex by the proteasome and/or a physical blockage of thecomplex's entry into the interior of the proteasome in-situ. These areindirect mechanisms of action in that the PR-39 protein is believed tointeract with the ubiquitinated-IκBα complex rather than affect theproteasome itself; and such indirect mechanisms exist concurrently withand are functional alternatives available on-demand in addition to anydirect action/inactivation mechanisms.

[0041] To initiate a demonstrable mechanism of action (indirect ordirect) and effect in-situ, the introduction of native PR-39 peptide (orone of its substituted forms or its shorter-length analogs or homologs)is the sole necessary prerequisite. Only the presence of sufficientPR-39 peptide (or any of its substituted or homologic equivalents)quantitatively to interact selectively with such proteasomes are aspresent intracellularly within viable cells is needed to invoke thePR-39 effect under both in-vivo conditions and in-vitro usecircumstances.

[0042] The methodology and means provided by the present invention forselectively inhibiting IκBα proteolysis, reducing NFκB activity, anddecreasing NFκB-dependent gene expression within viable cells istherefore directed at and focused upon the intracellular degradationcapability and function of proteasomes. Such selective inhibition and/ordisruption of proteasome-mediated protein degradation is achieved viathe introduction of native PR-39 peptide or a member of theshorter-length PR-39 derived oligopeptide family in any regimen oftreatment.

II. Proteasomes

[0043] The proteasome is a component of theubiquitin-proteasome-dependent proteolysis system. This system plays amajor role in the turnover of intracellular proteins, of misfoldedproteins, and in the selective degradation of key proteins. Controlledprotein degradation is an important and efficient way to removenonfunctional proteins and/or to regulate the activity of key proteins.Target proteins are selectively recognized by the ubiquitin system andsubsequently marked by covalent linkage of multiple molecules ofubiquitin, a small conserved protein. The polyubiquitinated proteins aredegraded by 26S proteasome. This complex, however, is composed of twolarge subcomplexes: the 20S proteasome constituting the proteolytic coreand the 19S regulatory complex which confers polyubiquitin binding andenergy dependence. A simplified scheme of the ubiquitin pathway isdepicted by Flow Scheme A below.

[0044] A substantial quantum of research has been conducted tounderstand the architecture, assembly, and molecular biology of theproteasome. Merely representative of scientific publications in thisfield are the following, the individual texts of which are expresslyincorporated by reference herein: Goldberg et al., Biol. Chem. 378:131-140 (1997); Tanaka, K., Biochem. Biophys. Res. Commun. 247: 537-541(1998); Baumeister et al., Cell 92: 367-380 (1998); Gerards et al., CMLS54: 253-262 (1998); Maurizi, M. R., Curr. Biol. 8: R453-R456 (1998);Rechsteiner et al., J. Biol. Chem. 268: 6065-6068 (1993); Gerards etal., J. Mol. Biol. 275: 113-121 (1998); Fenteany, G. and S. Schreiber,J. Biol. Chem. 273: 8545-8548 (1998); and Oikawa et al., Biochem.Biophys. Res. Commun. 246: 243-248 (1998).

[0045] The 20S Proteasome

[0046] The degrading component in ubiquitin-dependent protealysis is the26S proteasome. The catalytic core of this complex is the 20Sproteasome, which is highly conserved and can be found in eukaryotes,archaebacteria, and some eubacteria. In eukaryotes, the amount ofproteasomes can constitute up to 1% of the cell content, depending onthe average protein breakdown rates of the organ. Proteasomes arelocalized in the nucleus and the cytosol, sometimes colocalizing orassociating with the cytosketon. [See for example: Hilt, W. and D. H.Wolf, Trends Biochem. Sci. 21: 96-102 (1996); Ciechanover, A., Cell 79:13-21 (1994); Jentseh, S. and S. Schlenker, Cell 82: 881-884 (1995);Coux et al., Annu. Rev. Biochem. 65: 807-847 (1996); Dahlmann et al.,FEBS Lett. 251: 125-131 (1989); Tamura et al., Curr. Biol. 5: 766-774(1995); Machiels et al., Eur. J. Cell Biol. 66: 282-292 (1995);Scherrer, K. and F. Bey, Prog. Nucleic Acid Res. Mol. Biol. 49: 1-64(1994); and Gerards et al., CMLS 54: 253-262 (1998)].

[0047] The first description of a “cylinder-shaped” complex withproteasome-like features dates back to the late 1960s. The plethora ofnames given to it subsequently is a reflection of the problems that wereencountered over a period of two decades in trying to define itsbiochemical properties and cellular functions. Enzymological studiesrevealed an array of distinct proteolytic activities and led to aconsensus name, ‘multicatalytic proteinase’. This name, however, wassoon replaced by a new one, the ‘proteasome’ emphasizing its characteras a molecular machine.

[0048] At about the same time, it was found that the occurrence ofproteasomes was not restricted to eukaryotic cells. A compositionallysimpler, but structurally strikingly similar proteolytic complex wasfound in the archaeon Thermoplasma acidophilum, which later took apivotal role in elucidating the structure and enzymatic mechanism of theproteasome.

[0049] Nomenclature

[0050] The 20S proteasome was independently discovered by groups workingin different fields, and hence was given a variety of different names.In 1970, Scherrer and colleagues observed ring-shaped particles inribosome-free messenger RNA (mRNA) preparations [Sporh et al., Eur. J.Biochem. 17: 296-318 (1970)]. Subsequently, in 1979, DeMartino andGoldberg isolated a 700-kDa ‘neutral protease’ from rat liver[DeMartino, G. N. and A. L. Goldberg, J. Biol. Chem. 254: 3712-3715(1997)]. Then, in 1980 Wilk and Orlowski isolated a large proteasecomplex from the pituitary that possessed three different catalyticactivities. They called it multicatalytic protease [Wilk, S. and M.Orlowski, J. Neurochem. 35: 1172-1182 (1980); Wilk, S. and M. Orlowski,J. Neurochem. 40: 842-849 (1983)]. Later, Monaco and McDevittimmunoprecipitated complexes consisting of low molecular weight proteins(LMPs) with a possible role in antigen presentation [Monaco, J. J. andH. O. McDevitt, Nature 309: 797-799 (1984)]. Also, in 1984 this particlewas called prosome, referring to its presumed role in programming mRNAtranslation [Schmid et al., EMBO 3: 29-34 (1984)]. Altogether, thiscomplex has been given 21 different names in the literature. Since allparticles were shown to be identical the name ‘proteasome’ (which is nowgenerally accepted). was proposed first, referring to its proteolyticand particulate nature [Arrigo et al., Nature 331: 192-194 (1988);Faulkenburg et al., Nature 331: 190-192 (1988); Brown et al., Nature353: 355-357 (1991)].

[0051] Overall Characteristics and Properties

[0052] The 20S proteasome is the major cytosolic protease in eukaryoticcells and is the proteolytic component of the ubiquitin-dependentdegradative pathway. Proteasomes are also found in some, but not all,archaebacteria and eubacteria, and in eukaryotes. True proteasomes arecomposed of 28 subunits, 14 each of two different classes—non-catalyticalpha (α) and catalytically-active beta (β) subunits. The subunits arearranged in rings of seven subunits, all of a single type. The 20Sproteasome is a stack of four rings, two inner beta rings flanked by thealpha rings. The junction between the beta rings produces a remarkablestructural feature of proteasomes—an interior aqueous cavity largeenough to accommodate about 70 kDa of protein and accessible onlythrough narrow axial channels in the rings. The catalytic sites arelocated on the beta subunits within the aqueous cavity. Isolation of thecatalytic sites in this way, and the limited access via narrow channels,serves to compartmentalize proteolysis, allowing degradation of onlythose proteins that can be actively translocated into the interior ofthe proteasome.

[0053] Structure and Subunit Components

[0054] The 20S proteasome has a cylindrical or barrel-like structure,typically 14.8 nm in length and 11.3 nm in diameter. It is composed of28 subunits and arranged in four stacked rings, resulting in a molecularmass of about 700 kDa. This overall structural architecture is conservedfrom bacteria to man.

[0055] In eukaryotes, including humans, 14 different subunits, rangingfrom 21 kDa to 32 kDa, are present in the complex. Based on the sequencehomology with the T. acidophilum α- or β-subunit, the eukaryoticsubunits are divided into α-type and β-type, respectively [Zwicki etal., Biochemistry 31: 964-972 (1992); Heinemeyer et al., Biochemistry33: 12229-12237 (1994); Coux et al., Mol. Gen. Genet. 245: 769-780(1994)]. Table 1 shows some characteristics and alternative names of thesubunits of the human and yeast 20S proteasome using the older and thenew nomenclature proposed by Groll and coworkers [Groll et al., Nature386: 463-471 (1997)]. Immuno-electron microscopy (EM) studies alsorevealed that the eukaryotic α-type subunits reside in the outer ringsand the β-type subunits in the inner rings. Furthermore, these studiesindicated that in the eukaryotic 20S proteasome seven different subunitconstitute a ring, each subunit located at a defined position [Kopp etal., J. Mol. Biol. 229: 14-19 (1993); Kopp et al., J. Mol. Biol. 248:264-272 (1995); Schauer et al., J. Struct. Biol. 111: 135-147 (1993);Kopp et al., Proc Natl Acad Sci USA 94: 2939-2944 (1997)]. Therefore,the eukaryotic proteasome assembles as an α₁₋₇β₁₋₇β₁₋₇α₁₋₇ particle. Thetypical human structure and assembly is illustrated by Table 2. TABLE 1Nomenclature and molecular masses of proteasomal subunits SystematicMolecular mass of name Human gene Yeast gene human subunit (kDa) α1HsPROS27 HsIota C7 PRS2 27.4 α2 HsC3 Y7 25.9 α3 HsC9 Y13 29.5 α4 XAPC7HsC6 PRE6 27.9 α5 HsZeta PUP2 26.4 α6 HsPROS30 HsC2 PRE5 30.2 α7 HsC8 C1PRS1 28.4 β1 HsDelta Y PRE3 25.3 (21.9) β1i LMP2 23.2 (20.9) β2 Z PUP130.0 (24.5) β2i MECL1 28.9 (23.8) β3 HsC10-11 PUP3 22.9 β4 HsC7-1 PRE1C11 22.8 β5 MB1 X PRE2   nd (22.4) β5i LMP7 30.4 (21.2) β6 HsC5 C5 PRS326.5 (23.3) β7 HsBPROS26 HsN3 Pre4 29.2 (24.4)

[0056]

[0057] Proteolytic Activity

[0058] The first report on the multicatalytic properties of theproteasome stems from 1983, when three different proteolytic activitieswere distinguished: ‘trypsin-like’, ‘chymotrypsin-like’ and‘peptidylglutamyl-peptide hydrolase’ activity [Wilk, S. and M. Orlowski,J. Neurochem. 40: 842-849 (1983)]. These three proteasomal activitiesrefer to peptide bond cleavage at the carboxyl side of basic,hydrophobic and acidic amino acid residues, respectively. They wereidentified using short synthetic peptide substrates and are believed tobe catalyzed at independent sites—in part because the differentproteolytic activities respond differentially to various activators andinhibitors. With similar approaches, at least two additional proteolyticactivities have been recently described [Orlowski et al., Biochemistry32: 1563-1572 (1993); Orlowski, M., Biochemistry 29: 10289-10297 (1990);Rivett, A. J., Biochem. J. 291: 1-10 (1993)].

[0059] The Progressive Degradation of Protein Substrates

[0060] Recent studies have also revealed a fundamental new property ofthe proteasome that clearly distinguishes it from conventionalproteases: i.e., this particle degrades a protein substrate all the wayto small peptides, before attacking another protein substrate [Akopianet al., J. Biol. Chem. 272: 1791-1798 (1997)]. Because the proteasome'smultiple active sites are located in its central chamber and becausediffusion of a peptide substrate into this compartment must be a slowprocess, these particles function in a highly processive fashion; i.e.,they have mechanisms of action to bind tightly protein substrates and tomake multiple cleavages in the polypeptide before releasing the peptideproducts. Moreover, the ratio of new peptides generated to the number ofsubstrate molecules consumed is constant during the reaction. In otherwords, as peptides accumulated, they were not hydrolyzed further, evenduring prolonged incubations, where up to half of the substratemolecules were consumed. Equally important, the disappearance of thesesubstrate molecules coincided exactly with the appearance of smallpeptide products [Goldberg et al., Biol. Chem. 378: 131-140 (1997)].These observations, together with the finding that the pattern of theproducts is independent of time, established that processive degradationis a general feature of the 20S proteasome [Gerard et al., CMLS 54:253-262 (1998)].

[0061] The contribution of each individual active center and proteolyticactivity to the degradation of longer peptides and complete proteins ispresently unknown. Nevertheless, proteasomes are able to cleave behindmost amino acids in a protein. Thus, the 20S proteasome is in fact anonspecific endopeptidase. In addition, however, the generated(degraded) peptides fall into a rather narrow size range of 6 to 10amino acids in length, demonstrating the existence of a kind of‘molecular ruler’. The average length of the degradation products istypically 7 to 8 amino acids; this finding is in agreement with thedistance between the active sites in the proteasome. Similar nonspecificendopeptidase activity and size distribution of degration products fromwhole proteins was observed for proteasomes generally and by proteasomesof human origin in particular.

[0062] Other features of the 20S proteasome degradation are also unique.While unfolded peptides are usually digested, most native proteins areresistant to proteolytic degradation by the 20S proteasome in vitro.However, denaturation of the substrate protein by oxidation or reductionof disulphide bridges can render it accessible to degradation byproteasomes. Also, small gold particles with a diameter of 2 nmcontaining unfolded substrate cannot enter the proteasome. Thesecharacteristics show that a relatively narrow opening controls access tothe inner proteolytic compartment of the proteasome.

III. IκBα Protein and NFκB Transcription Factor

[0063] Regulation of the immune and inflammatory responses requires theactivation of specific sets of genes by a variety of extracellularsignals. These signals include mitogens (e.g., lipopolysaccharide andphorbol myristate acetate), cytokines (e.g., tumor necrosis factor α andinterleukin-1β), viral proteins (e.g., the Tax protein of type 1 human Tcell leukemia virus), antigens, phosphatase inhibitors (e.g., okadaicacid and calyculin A), and ultraviolet light.

[0064] The Rel/NFκB family of transcriptional activator proteins playsan essential role in the signal transduction pathways that link thesesignals to gene activation [Siebenlist et al., Annu. Rev. Cell Biol. 10:405-455 (1994); Thanos and Maniatis, Cell 80: 529-532 (1995); Verma etal., Gene Dev. 9: 2723-2735 (1995)]. NFκB (p50/RelA[p65]) and otherheterodimeric Rel family proteins are sequestered in the cytoplasmthrough their association with members of the IκB family of inhibitorproteins. In the case of IκBα, stimulation of cells leads to rapidphosphorylation and degradation of the inhibitor protein. Consequently,NFκB is released and translocates into the nucleus, where it activatesthe expression of target genes.

[0065] NFκB itself is an inducible transcriptional factor whichregulates many biologically important processes, such as stress,inflammation, development and immune response [May, M. J. and S. Ghosh,Immunol. Today 19: 80-88 (1998)]. The prototypical inducible NFκBcomplex is a heterodimer of the Rel protein family, consisting of p50and p65 (also called Rel A) [Baeverle, P. A. and D. Baltimore, Cell 87:13-20 (1996); Baldwin, A. S., Anno. Rev. Immunol. 14: 649-681 (1996)].In the cytoplasmic compartment of many types of cells, NFκB normallyexists in an inactive form due to association of its protein inhibitor,termed IκB. IκB prevents the transport of NFκB into the nucleus bymasking the nuclear localization signal (NLS) of NFκB [Finco, T. S. andA. S. Baldwin, Immunity 3: 253-272 (1995)]. This action is mediated bythe multiple, tandemly-repeated ankyrin repeats present on IκB, whichare thought to interact with NFκB. To date, there are 9 members in thestructurally and functionally related IκB protein family: IκBα, IκBβ,IκBε, IκBγ (-1,-2), Bcl-3, p100, and p105, have been identified[Whiteside, S. T. and A. Israel, Cancer Biol. 8: 75-82 (1997)].

[0066] It is recognized that IκBα binding with NFκB will causeinactivation of NFκB function. However, phosphorylation of IκBα per seis not sufficient to dissociate NFκB from the latent complex [Palombellaet al., Mol. Cell. Biol. 15: 1294-1301 (1995)]. Rather, phosphorylationtriggers the degradation of IκBα [Brockman et al., Mol. Cell. Biol. 15:2809-2818 (1995); Brown et al., Science 267: 1485-1490 (1995)].

[0067] Various external stimuli, such as cyotkines including alpha-typetumor necrosis factor (TNFα), viral infection, T-cell and B-cellmitogens, and UV-stress, initiate the immediate removal of IκB from theIκB-(NFκB) complex [May, M. J. and S. Ghosh, Immunol. Today 19: 80-85(1998); Finco, T. S. and A. S. Baldwin, Immunity 3: 253-272 (1995)]. Thetremendous progress in delineating the molecular mechanisms of the NFκBsignaling pathway has revealed that phosphorylation of 2 serine residues(Ser-32 and Ser-36) near the N-terminus of IκBα is essential fortargeting IκBα for signal-promoted destruction [Traenckner et al., EMBOJ. 14: 2876-2883 (1995); Chen et al., Genes Dev. 9: 1586-1597 (1995);DiDonato et al., Mol. Cell. Biol. 16: 1295-1304 (1996)]. This specificphosphorylation of IκBα is catalyzed by an unusually large,multi-protein kinase complex, termed IκB kinase (abbreviated IKK), withan apparent molecular mass of 700-900 kDa. The phosphorylation of IκBαby an IKK complex is necessary for its poly-ubiquitination at residuesLys-21 and Lys-22 [Scherer et al., Proc. Natl. Acad. Sci. USA 92:11259-11263 (1995); Baldi et al., J. Biol. Chem. 271: 376-379 (1996);Rodriguez et al., Oncogene 12: 2425-2435 (1996); Spencer et al., GenesDev. 13: 284-294 (1999)]. The ubiquitin (Ub)-proteasome system thenplays the next indispensable role for down-regulating IκBα at thephysiologic level [Traenckner et al., EMBO J. 13: 5433-5441 (1994); Chenet al., Cell 84: 853-862 (1996)].

[0068] Identification of IKB Activity

[0069] When first discovered, NFκB was thought to be a transcriptionfactor specific to β cells. However, by treating cytosolic extracts fromother cell types with a mixture of detergents the presence of latentNFκB DNA-binding activity in the cytoplasm was detected. Furthermore,cytoplasmic extracts were found to contain activities that could inhibitNFκB DNA-binding activity. Therefore, the existence of a cytoplasmicinhibitor of NFκB activity, I kappa B (IκB), which would becomeinactivated upon induction of NFκB activity was proposed [Baeuerle, P.A. and D. Baltimore, Cell 53: 211-217 (1988)]. (IκB is also able todissociate preformed NFκB/DNA complexes, leading to the hypothesis of arole for IκB in the nucleus).

[0070] IκB activity was found to be biochemically purifiable as twoimmunologically distinct activities, α and β, which differ slightly intheir molecular weights (37 kDa and 43 kDa, respectively) as well astheir mechanism of inactivation: whereas purified IκBα could beinactivated by treatment with various purified kinases, IκBβ could beinactivated by phosphatase treatment [Link et al., J. Biol. Chem. 267:239-246 (1992)]. Recently, a third cytoplasmic IκB protein, IκBε, whichcontrols the activity of a subset of Rel/NFκB transcription complexes,has also been identified [Whiteside et al., EMBO J. 16: 1413-1426(1997)]. cDNAs encoding two other IκB-like proteins, IκBDL and IκBR,have also been isolated, although their relevance to the regulation ofNFκB activity remains uncertain [Albertella et al., Hum. Mol. Genet. 3:793-799 (1994); Ray et al., J. Biol. Chem. 270: 10680-10685 (1995)].IκBR is a 52 kDa molecule that is preferentially expressed in epithelialcells.

[0071] In addition, the p105 and p100 precursor proteins of the p50 andp52 DNA-binding subunits of NFκB exhibit the properties of IκBmolecules, in that they are able to sequester NFκB proteins (in the formof monomers) in the cytoplasm [Rice et al., Cell 7: 243-253 (1992)]. TheNFκB proteins sequestered by p105 or p100 are not activatable bydetergents in vitro.

[0072] The C-terminal portion of the p105 protein, IκB-γ, has also beenfound to exist as a 70 kDa protein in its own right in certain celltypes, synthesized from an internal promoter of the same gene [Inoue etal., Cell 68: 1109-1120 (1992)], although the function of IκB-γ has onlybeen found in mouse cells to date. In addition, alternative splicing ofIκB-γ has been shown to generate two smaller molecules, IκB-γ-1 (63 kDa)and Iκb-γ-2 (55 kDa) which can both inhibit NFκB [Grumont, R. J. and S.Gerondakis, Proc. Natl. Acad. Sci. USA 91: 4367-4371 (1994)]. By analogyto IκB-γ, a C-terminal form of p100 has been hypothesized to exist(IκB-δ) [Dobrzanski et al., Oncogene 10: 1003-1007 (1995)], but IκB-δhas not been rigorously identified in vivo.

[0073] Finally, there exists a nuclear IκB protein, Bcl-3, which cancomplex with specific dimers (such as p522 and p502) and transactivateκB-dependent transcription in a manner that depends on the type of NFκBcomplex present [Bours et al., Cell 72: 729-739 (1993)].

[0074] Thus, there are today nine different vertebrate IκB proteins thathave been identified.

[0075] Structure of the IκB Molecules

[0076] All proteins in the IκB family contain multiple copies of astructural motif known as the ankyrin repeat, which is important forprotein-protein interactions (see Table 3). IκB-α, -β and -ε contain sixankyrin repeats that are necessary for interaction with NFκB.Mutagenesis studies have shown that the ankryin repeats of IκBα interactwith NFκB causing NFκB to remain in the cytoplasm by masking the nuclearlocalization sequence (NLS) situated in the C-terminal region of the Relhomology domain (RHD) [Beg et al., Genes Dev. 6: 1899-1913 (1992);Ganchi et al., Mol. Biol. Cell. 3: 1339-1352 (1992)].

[0077] The different IκB molecules show specificity for binding andinhibiting various Rel/NFκB complexes. For example, IκB-α and IκB-βinteract with heterodimers of p50 or p52 complexed with RelA or c-Rel,as well as homodimers and heterodimers of RelA and c-Rel. In contrast,IκB-ε appears to be complexed almost exclusively with dimers thatcontain only RelA and/or c-Rel proteins. These dimers differ fromclassical NFκB complexes in their DNA-binding specificity, and thusregulate the activity of different subsets of NFκB-dependent genes.

[0078] In addition, IκB-β, IκB-β and IκB-ε contain near their N-terminala pair of serine residues separated by three amino acids that areessential for the regulation of their ability to inhibit NFκB induction.Finally, IκB-αand IκB-β contain a C-terminal region rich in proline,glutamine, asparate, serine and threonine residues (a so-called PESTdomain). The PEST domain is a site for phosphorylation and has beenimplicated in regulating the stability of IκB as well as playing a rolein the ability of IκB to inhibit DNA-binding by Rel/NFκB complexes,perhaps via direct interaction with the DNA-binding domain.

[0079] Nuclear Functions of IKB

[0080] IκBα, IκBβ and IκBε molecules differ in their response toinducers of NFκB. IκBα is rapidly degraded in response to all inducersof NFκB thus far tested; and is subsequently resynthesized in anNFκB-dependent manner. This latter property (coupled with theobservation that IκBα is able to remove preformed NFκB complexes fromtheir cognate binding sites) led to the hypothesis that IκBα plays arole in ensuring the transient nature of the NFκB response. Furthermore,in the case of negative regulation of cytokine expression byglucocorticoids, the induced synthesis of IκBα leads to nucleartranslocation of IκBα whereupon it can inhibit the binding of NFκB toits target sites [Arenzana-Seisdedos et al., Mol. Cell. Biol. 15:2689-2696 (1995)]. Nuclear IκB would bind to and remove NFκB complexesfrom DNA, whereupon a nuclear export sequence (NES) in IκBα (exposedupon binding to NFκB) results in a net explusion of NFκB/IκB complexesfrom the nucleus [Fritz, C. C. and M. R. Green, Curr. Biol. 6: 848-854(1996)].

[0081] Whereas IκBα is rapidly degraded in response to inducers of NFκBactivity, IκBβ and IκBε are degraded more slowly. In the case of IκBβ,it has been proposed that this protein regulates the persistent responseof a subset of NFκB inducers. The NFκB molecules regulated by IκBβescape control by IκBα by a novel mechanism: in unstimulated cells, NFκBis associated with a hyperphosphorylated form of IκBβ while instimulated cells NFκB is found in complexes that contain ahypophosphorylated form of IκBβ. These complexes have an exposed NLS andare insensitive to IκBα inhibition. Furthermore, the ternary NFκB/IκBβcomplexes can be found in the nucleus of stimulated cells and are ableto bind to DNA. It has therefore been proposed that thehypophosphorylated form of IκBβ acts as a chaperone to protect NFκB fromIκBα, allowing NFκB to translocate to the nucleus, whereupon IκBβ iseither degraded, or is dissociated from NFκB following binding of NFκBto DNA.

[0082] IκBε, which is also degraded slowly but resynthesized shortlyafterwards, is thought to control a third type of NFκB response, onethat is slow, but transient in nature. Although the expression of IκBεis upregulated following induction of NFκB, and while it exists asmultiple phospho-isoforms in unstimulated cells, IκBε has yet to bedetected in the nucleus and does not appear to contain an NES.

[0083] Inducible Degradation of IκB

[0084] While IκBα, IκBβ and IκBε each have distinct functions, theirdegradation appears to involve a similar mechanism. Each IκB contains inits N-terminal region a pair of serine residues that lie in similarsequence contexts. In the case of IκBα, these serine residues (aminoacids 32 and 36 in human IκBα) become phosphorylated by aserine-specific kinase following stimulation. This phosphorylation,which does not dissociate IκBα from NFκB, renders IκBα a substrate forubiquintination (primarily at lysine residues 21 and 22) which in turntargets the protein for degradation by the 26S proteasome. Although IκBβand IκBε contain lysine residues in similar positions, these lysines arenot absolutely required for ubiquitination to occur. The C-terminal PESTdomain also appears important for signal-induced ubiquitination andproteolysis.

[0085] Ubiquitin and proteasomes are the principal components of anenergy-dependent proteolytic system in eukaryotic cells for such as IκBαdestruction. Selective destruction of cellular proteins by this systemoccurs by two sequential processes. The first is selective marking ofcandidate IκB proteins for degradation by the covalent attachment of apoly-Ub chain [Aershko, A. and A. Ciechanover, Annu. Rev. Biochem. 67:425-479 (1998)]. The second process is proteolytic attack ofpoly-ubiquitinated IκB proteins by the 26S proteasome, a eukaryoticATP-dependent 2-MDa protease complex [Coux et al., Annu. Rev. Biochem.65: 801-847 (1996); Baumeister et al., Cell 92: 367-380 (1998); andTanaka, K. and T. Chiba, Genes Cells 3: 485-498 (1998)].

[0086] The covalent attachment of Ub through its C-terminal Gly residueto the ε-NH₂ group of the Lys residue on substrate proteins is known tobe mediated by a cascade of three enzymes, designated E1(Ub-activating), E2 (Ub-conjugating), and E3 (Ub-ligating) [Kroll etal., J. Biol. Chem. 274: 7941-7945 (1999); Suzuki et al., Biochem.Biophys. Res. Comm. 256: 121-126 (1999); Chen et al., Cell 84: 853-862(1996); Yaron et al., Nature 396: 590-594 (1998)]. A poly-Ub chain isformed by linking the C-terminus of one Ub to a Lys residue withinanother Ub. The resultant poly-Ub chain acts as a degradation signal forproteolytic attack by the 26S proteasome.

[0087] In this pathway, the IκBα protein targeted for degradation isfirst modified by covalent attachment of Ub, a highly conservedpolypeptide of 76 amino acids. Ubiquination is a three-step process.First, Ub is activated by a Ub-activating enzyme (E1); the activated Ubis then transferred to a Ub carrier protein (E2, also referred to asUb-conjugating enzyme [Ubcl]); finally, Ub is conjugated to a proteinsubstrate by forming an isopeptide bond between the C-terminal glycineresidue of Ub and the ε-amino group of one or more lysine residues ofthe protein substrate. This conjugate step often requires a Ub proteinligase (E3). Multiple molecules of Ub can be ligated to a proteinsubstrate to form multi-Ub chains. These are then recognized by a largeATP-dependent protease, the 26S proteasome, composed of a 20S catalyticcore and a 19S regulatory complex.

[0088] Ubiquitination of IκBα is regulated by signal-inducedphosphorylation at two specific residues, serine residues 32 and 36.Single amino acid substitutions of one or both of these residues abolishthe signal-induced phosphorylation and degradation of IκBα in vivo. Thesame mutations also abolish the okadaic acid-induced phosphorylation andubiquitination of IκBα in vitro. Relatively little is known about thesignal transduction pathways and the kinase(s) responsible for thesite-specific phosphorylation of IκBα. Mutants of IκBα lacking S32 andS36 are resistent to induced phosphorylation by a variety of stimuli,suggesting that different signal transduction pathways converge on aspecific kinase or kinases.

IV. The PR-39 Oligopeptide Collective

[0089] Native PR-39 peptide is a substance belonging to the cathelinfamily of proteins; the mature peptide is 39 amino acids in length inthe naturally occurring state; and the peptide is able to exert avariety of activities and cause different cellular outcomes. Althoughfirst identified as a membrane permeating antibacterial peptide found inthe intestine of pigs [Agerberth et al., Eur. J. Biochem. 202: 849-854(1991)], this peptide was subsequently isolated from wounds where itcould simultaneously reduce infection and influence the action of growthfactors, matrix components, and other cellular effectors involved inwound repair [Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039(1994); Gallo et al., J. Invest. Dermatel. 104: 555 (1995)]. Thestructure and membrane interactions of native PR-39 peptide have alsobeen elucidated [Cariaux et al., Eur. J. Biochem. 224: 1019-1027 (1994)]and the complete amino acid sequences of native PR-39 peptide and itsvarious substituted forms have been reported [PCT Publication No. WO92/22578 published 23 Dec. 1992].

[0090] More recently, the native PR-39 peptide was shown to possess asyndecan-inducing activity in furtherance of its wound healingcapabilities; and while renamed a “synducin”, was shown to inducecellular production of two specific proteoglycans, syndecan-1 andsyndecan-4, within living mesenchymal cells [U.S. Pat. No. 5,654,273].Overall, native PR-39 peptide has been shown to play a role in severalinflammatory events including wound healing and myocardial infarction[Gallo et al., Proc. Natl. Acad. Sci. USA 91: 11035-11039 (1994); Li etal., Circ. Res. 81: 785-796 (1997)]; and the native peptide has beenshown to be taken up rapidly by a number of different cell typesincluding meschymal cells and endothelial cells [Chan, Y. R. and R. L.Gallo, J. Biol. Chem. 273: 28978-28985 (1998)].

[0091] The PR-39 Peptide Grouping

[0092] Native PR-39 peptide is composed of the 39 amino acid sequenceshown below (and also by Table 4). PR-39:Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro-Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-Pro-Pro-Gly-Phe-Pro-Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro

[0093] As conventionally known and reported [see for example, U.S. Pat.No. 5,654,273], the specific peptide can be substituted usingconservative substitutions of amino acids having the same orfunctionally equivalent charge and structure, except for the requiredamino acid sequence “Arg-Arg-Arg” at the N-terminus and the intermediateamino acid sequences “Pro-Pro-X-X-Pro-Pro-X-X-Pro” and“Pro-Pro-X-X-X-Pro-Pro-X-X-Pro” where X can be substituted freely usingany amino acid. Thus, all of the preferred substituted amino acidsequences are of about the same size and each differ from the nativePR-39 peptide sequence only by substitutions in the intermediateportions of the structure.

[0094] The PR-39 Derived Oligopeptide Family

[0095] In addition to the conventionally known native PR-39 peptideamino acid residue sequence and its readily recognizable substitutedforms as described above, an entirely novel and unforeseen family ofPR-39 derived oligopeptide structures is provided by the presentinvention for use. This previously unknown family of PR-39 derivedoligopeptides is constituted of members which individually will cause aselective inhibition of proteasome-mediated degradation of peptidesin-situ after introduction intracellularly to a viable cell.

[0096] Each member of this PR-39 derived oligopeptide family presentscharacteristics and properties which are commonly shared among theentire membership. These include the following:

[0097] (i) each peptide sequence is less than 39 amino acid residues inlength in every embodiment, and preferably is less than 20 residues insize in the best mode;

[0098] (ii) each short-length peptide sequence is at least partiallyhomologous (or analogous) with the N-terminal amino acid residues of thenative PR-39 peptide, and preferably is completely identical or markedlysimilar to the N-terminal end residues of the native PR-39 peptide;

[0099] (iii) each short-length peptide is able to interact in-situ withat least a part of such proteasomes as are present within the cytoplasmof the cell; and

[0100] (iv) each short-length peptide sequence is able to alter markedlythe proteolytic activity of proteasomes such that a selective increasedexpression of specific proteins (such as IκBα and HIF-1α) occursin-situ.

[0101] Merely as illustrative examples and preferred embodiments of thebroad membership constituting this PR-39 derived oligopeptide family,the members comprising 15, 11 and 8 amino acid residues respectively inlength are presented below as the PR15, PR11, and PR8 entitiesrespectively. Note also that some substituted analogs of these shorterlength entities provide biologically active formats. For example, theaddition of an amino group in N-terminal opposition or the addition ofan acetyl group in the C-terminal position stabilizes and enhancesactivity of PR11. For comparison purposes only, the complete amino acidsequence of the native PR-39 peptide is presented as well.1   2   3   4   5   6   7   8   9   10  11  12  13 PR-39:Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-14  15  16  17  18  19  20  21  22  23  24  25  26  Pro-Pro-Pro-Phe-Phe-Pro-Pro-Arg-Leu-Pro-Pro-Arg-Ile-27  28  29  30  31  32  33  34  35  36  37  38  39Pro-Pro-Gly-Phe-Pro-Pro-Arg-Phe-Pro-Pro-Arg-Phe-Pro PR-15:1   2   3   4   5   6   7   8   9   10  11  12  13  14  15Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro PR-11:1   2   3   4   5   6   7   8   9   10  11  Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg PR-8:1   2   3   4   5   6   7   8 Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr

[0102] The PR-39 Oligopeptide Collective

[0103] Terminology and nomenclature often pose problems for the readeras to what precisely is meant. Accordingly, for definitional purposes,avoidance of ambiguities, and clarity of understanding, the followingterms and titles will be employed herein. The term “PR-39 peptidesgrouping” includes by definition the native PR-39 structure and allsubstituted forms conventionally known of the naturally occurring 39length amino acid sequence. In distinction, the term “PR-39 derivedoligopeptide family” and its members includes by definition all thepreviously unknown shorter-length homologs and analogs as well assubstituted forms of the native PR-39 structure as described above.Finally, the umbrella term and category title “PR-39 oligopeptidecollective” includes by definition both the ‘PR-39 peptide grouping’ aswell as the ‘PR-39 derived oligopeptide family’ members, and identifiesany and all individual structures falling into either of the two subsetcategories. TABLE 4 (1) GENERAL INFORMATION:     (i) APPLICANT:Children's Medical Center Corporaton    (ii) TITLE OF INVENTION:Synducin Mediated Modulation of Tissue Repair    (iii) NI.ThiBBR OFSEQUENCES: 4     (iv) CORRESPONDENCE ADDRESS:           (A) ADDRESSEE:Patree L. Pabst           (B) STREET: 2800 One Atlantic Center                      1201 West Peachtree           (C) CITY: Atlanta          (D) STATE: Georgia           (E) COUNTRY: USA           (F)ZIP: 30309-3450     (v) COMPUTER READABLE FORM:          (A) MEDIUMTYPE: Floppy disk          (B) COMPUTER: IBM PC compatible          (C)OPERATING SYSTEN: PC-DOS/MS-DOS          (D) SOFTWARE: PatentIn Release#1.0, Version #1.25     (ix) TELECOMMUNICATION INFORNATION:          (A)TELEPHONE: (404)-873-8794          (B) TELEFAX: (404)-815-8795 (2)INFORMATION FOR SEQ ID NO:1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 39 amino acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: peptide    (iii)HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO      (x) PUBLICATIONINFORMATION:           (A) AUTHORS: Lee, Joug-Youn                       Boman, Hans G.                        Mutt,Viktor                        Jornvall, Hans           (B) TITLE: NovelPolypeptides And Their Use           (C) JOURNAL: PCT WO 92/22578          (D) DATE: 12/23/92           (K) RELEVANT RESIDUES IN SEQ IDNO:1: FROM 1 TO 39     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:      ArgArg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro     1               5               10 15      Phe Phe Pro Pro Arg LeuPro Pro Arg Ile Pro Pro Gly Phe Pro Pro                 20                  25                  30      Arg PhePro Pro Arg Phe Pro              35

[0104] Synthesis

[0105] The PR-39 peptide can be synthesized using standard amino acidsynthetic techniques. An example is the conventionally used solid phasesynthesis [Merrifield, J., J. Am. Chem. Soc. 85: 2149 (1964)] describedin U.S. Pat. No. 4,244,946, wherein a protected alpha-amino acid iscoupled to a suitable resin, to initiate synthesis of a peptide startingfrom the C-terminus of the peptide. Other methods of peptide synthesisare described in U.S. Pat. Nos. 4,305,872 and 4,316,891, the teachingsof which are incorporated herein. These methods can be used tosynthesize peptides having identity with the native PR-39 peptide aminoacid sequence described herein, or to construct desired substitutions oradditions of specific amino acids, which can be screened for content andevaluated for activity. PR-39 can also be commercially obtained fromMagainin, Inc. (Plymouth Meeting, Pa.).

[0106] Pharmaceutical Formats

[0107] After synthesis or purchase, the PR-39 peptides (as a family ofhomologs and analogs with substituted amino acid residues) can beintroduced as a pep tide-containing preparation in a pharmaceuticallyacceptable format.

[0108] The PR-39 can be administered and introduced in-vivosystemically, topically, or locally. The peptide can be administered asthe peptide or as a pharmaceutically acceptable acid- or base-additionsalt, formed by reaction with an inorganic acid (such as hydrochloricacid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, and phosphoric acid); or with an organic acid (such asformic acid, acetic acid, propionic acid, glycolic acid, lactic acid,pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, andfumaric acid); or by reaction with an inorganic base (such as sodiumhydroxide, ammonium hydroxide, potassium hydroxide); or with an organicbase (such as mono-, di-, trialkyl and aryl amines and substitutedethanolamines).

[0109] PR-39 peptide and any of the PR-39 derived oligopeptide familymembers may also be conjugated to sugars, lipids, other polypeptides,nucleic acids and PNA; and function in-situ as a conjugate or bereleased locally after reaching a targeted tissue or organ. The PR-39family of peptides may also be linked to targeting compounds forattachment in-situ to a specific cell type, tissue or organ.

V. Means for Introduction of PR-39 Peptide and/or its Shorter-LengthDerived Homologs

[0110] DNA Fragments and Expression Vectors

[0111] A variety of means and methods are conventionally known andpresently available to the user or practitioner of the present inventionin order to introduce PR-39 peptide (or a derived oligopeptide familymember) to living cells and tissues. One desirable means uses a preparedDNA sequence fragment encoding the PR-39 peptide (or a shorter-lengthhomolog) in a suitable vector as the means of introduction to theintended target in-situ. These means for delivery envision and includein-vivo use circumstances; ex-vivo specimens and conditions; andin-vitro cultures. In addition, the present invention intends andexpects that the prepared DNA sequence fragment coding for PR-39 peptide(or shorter-length homologs) has been inserted in a suitable expressionvector and will be used in a route of administration for delivery toliving tissues comprising endothelial cells, and typically vascularendothelial cells which constitute the basal layer of cells withincapillaries and blood vessels generally. Clearly, the cell recipientsthemselves are thus eukarytoic in origin, typically mammalian cells fromhuman and animal sources; and most typically would include the higherorders of mammals such as humans and domesticated mammalian animals keptas pets or sources of food intended for future consumption. Accordingly,the range of animals includes all domesticated varieties involved innutrition including cattle, sheep, pigs and the like; as well as thoseanimals typically used as pets or raised for commercial purposesincluding horses, dogs, cats, and other living mammals typically livingwith and around humans.

[0112] Clearly, the expression vectors must be suitable for transfectionof endothelial cells in living tissues of mammalian origin and thus becompatible with that type and condition of cells under both in-vivoand/or in-vitro conditions. The expression vectors thus typicallyinclude plasmids and viruses as expression vectors.

[0113] Also, both the plasmid based vectors and the viral expressionvectors constitute conventionally known means and methods ofintroduction which are conventionally recognized today as “gene therapy”modes of delivery. However, this overall approach is not the only meansand method of delivery available for the present invention.

[0114] Direct Introduction of Previously Synthesized PR-39 Peptides or aPR-39 Derived Oligopeptide Family Member

[0115] PR-39 peptide or an oligopeptide family member can be introduceddirectly as a synthesized compound to living cells and tissues via arange of different delivery means. These include the following.

[0116] 1. Intracoronary delivery is accomplished using catheter-baseddeliveries of synthesized PR-39 peptide (or homolog member) suspended ina suitable buffer (such as saline) which can be injected locally (i.e.,by injecting into the myocardium through the vessel wall) in thecoronary artery using a suitable local delivery catheter such as a 10 mmInfusaSleeve catheter (Local Med, Palo Alto, Calif.) loaded over a 3.0mm×20 mm angioplasty balloon, delivered over a 0.014 inch angioplastyguidewire. Delivery is typically accomplished by first inflating theangioplasty balloon to 30 psi, and then delivering the protein throughthe local delivery catheter at 80 psi over 30 seconds (this can bemodified to suit the delivery catheter).

[0117] 2. Intracoronary bolus infusion of PR-39 peptide (or ashort-length homolog) synthesized previously can be accomplished by amanual injection of the substance through an Ultrafuse-X dual lumencatheter (SciMed, Minneapolis, Minn.) or another suitable device intoproximal orifices of coronary arteries over 10 minutes.

[0118] 3. Pericardial delivery of synthesized PR-39 peptide (or ashorter-length homolog) is typically accomplished by instillation of thepeptide-containing solution into the pericardial sac. The pericardium isaccessed via a right atrial puncture, transthoracic puncture or via adirect surgical approach. Once the access is established, the peptidematerial is infused into the pericardial cavity and the catheter iswithdrawn. Alternatively, the delivery is accomplished via the aid ofslow-release polymers such as heparin-alginate or ethylene vinyl acetate(EVAc). In both cases, once the PR-39 peptide (or homolog) is integratedinto the polymer, the desired amount of PR-39/polymer is inserted underthe epicardial fat or secured to the myocardial surface using, forexample, sutures. In addition, the PR-39/polymer can be positioned alongthe adventitial surface of coronary vessels.

[0119] 4. Intramyocardial delivery of synthesized PR-39 peptide (or ashorter-length homolog) can be accomplished either under direct visionfollowing thoracotomy or using thoracoscope or via a catheter. In eithercase, the peptide containing solution is injected using a syringe orother suitable device directly into the myocardium. Up to 2 cc of volumecan be injected into any given spot and multiple locations (up to 30injections) can be done in each patient. Catheter-based injections arecarried out under fluoroscopic, ultrasound or Biosense NOGA guidance. Inall cases after catheter introduction into the left ventricle thedesired area of the myocardium is injected using a catheter that allowsfor controlled local delivery of the material.

[0120] Pharmaceutical Carriers of PR-39 Peptides or a PR-39 DerivedOligopeptide Family Member

[0121] A range of suitable pharmaceutical carriers and vehicles areknown conventionally to those skilled in the art. Thus, for parenteraladministration, the compound will typically be dissolved or suspended insterile water or saline.

[0122] For enteral administration, the PR-39 peptide or homologousoligopeptide of choice will be typically incorporated into an inertcarrier in tablet, liquid, or capsular form. Some suitable carriers arestarches and sugars; and often include lubricants, flavorings, binders,and other materials desirable in tablet making procedures.

[0123] The PR-39 peptide and oligopeptide family of compounds can alsobe administered topically by application of a solution, cream, gel, orpolymeric material (for example, a Pluronic™, BASF).

[0124] As an alternative, the chosen peptide can be administered inliposomes or microspheres (or microparticles), which can be injected forlocal or systemic delivery. Methods for preparing liposomes andmicrospheres for administration to a patient are conventionally known tothose skilled in the art. For example, U.S. Pat. No. 4,789,734 describesmethods for encapsulating biological materials in liposomes.Essentially, the material is dissolved in an aqueous solution, theappropriate phospholipids and lipids added, along with surfactants ifrequired, and the material dialyzed or sonicated, as necessary. Seealso, G. Gregoriadis, Chapter 14, “Liposomes”, Drug Carriers in Biologyand Medicine, chap. 14, pp. 287-341 (1979). Microspheres formed ofpolymers or proteins are well known to those skilled in the art, and canbe tailored for passage through the gastrointestinal tract directly intothe bloodstream. Alternatively, the compound can be incorporated and themicrospheres, or composite of microspheres, implanted from days tomonths. See, for example, U.S. Pat. Nos. 4,906,474; 4,925,673; and3,625,214.

[0125] Examplary Introductions and Preferred Routes of Administration

[0126] A variety of approaches, routes of administration, and deliverymethods have been identified herein and are available for introductionof PR-39 peptide and the derived family of oligopeptides. It isenvisioned, however, that a majority of the approaches and routes ofadministration described herein will be medical applications andspecific clinical approaches intended for use with individual humanpatients having specified medical problems and diagnosed pathologies. Itis expected, accordingly, that the reader is familiar generally with thetypical clinical human problem, pathology, and medical conditionsdescribed herein; and therefore will be able to follow and easilyunderstand the nature of the intervention clinically using the presentinvention and the intended outcome and result of the clinicaltreatment—particularly as pertains to the stimulation of angiogenesisunder in-vivo treatment conditions. A representative listing ofpreferred clinical approaches is given by Table 5 below. TABLE 5Preferred Routes of Administration Catheter-based (intracoronary)injections and infusions; Direct myocardial injection (intramyocardialguided); Direct myocardial injection (direct vision-epicardial-openchest or under thorascope guidance); Local intravascular delivery; Localtransvascular delivery; Perivascular delivery; Liposome-based delivery;Delivery in association with receptor-specific peptides; Oral delivery;In instances of peripheral vascular disease: intramuscular injection,intraarterial injection and/or infusion, perivascular delivery with orwithout sustained-release polymer.

VI. Illustrative Applications for the Methodology

[0127] a. Inhibition of ischemia-reperfusion injury. Restoration ofblood flow to the myocardium in the setting of acute myocardialinfarction leads to further damage to ischemic but still live heartmuscle due to an influx of inflammatory blood cells that is mediated byNFκB-activated expression of endothelial adhesion molecules ICAM-1 andVCAM-1. Infusion of PR39 prior to restoration of flow blocks this effectdue to inhibition of NFκB activity and effectively protects from furthermyocardial injury. Data in mice show that infusion of PR39 reducesinfarct size by 60% by this mechanism.

[0128] b. Inhibition of chemical pancreatitis. Tissue injury in thepancreas due to chemical or hormonal injury is one of the more commoncauses of acute pancreatitis. This is largely mediated by NFκB.Pre-treatment with PR39 blocks this injury.

[0129] c. PR39 will block any other injury activated NFκB-dependent genetranscription and thus can be used to block the development of skinwounds, ulcers (stomach, duodenum), etc.

VII. Experiments and Empirical Data

[0130] To demonstrate the merits and value of the present invention, aseries of planned experiments and empirical data are presented below. Itwill be expressly understood, however, that the experiments describedand the results provided are merely the best evidence of the subjectmatter as a whole which is the invention; and that the empirical data,while limited in content, is only illustrative of the scope of theinvention envisioned and claimed.

Experimental Series Methods and Materials

[0131] Cell Culture Studies

[0132] EVC304 cells (ATCC) were cultured in M199 medium supplementedwith 10% fetal bovine serum (FBS) and 10 μg/ml penicillin/streptomycin.U937 cells (courtesy Dr. J. Chang, BIDMC, Boston) were cultured inRPM1640 medium supplemented with 10% FBS and 100 μg/mlpenicillin/streptomycin.

[0133] Yeast Two-Hybrid Screening

[0134] Two-hybrid screening was done using Clontech MATCHMAKER GAL4System 2 (Clontech). cDNA of porcine PR39 peptide corresponding to the4th exon (amino acids 131 to 169) was subcloned into pAS2-1 vector asbait. Mouse embryo 3T3 MATCHMAKER cDNA library (Clontech) was screenedin the yeast Y190 strain. Plasmids from HIS3/LacZ positive clones weresequenced and co-transformed with bait plasmid back into the Y190 strainto confirm the interaction.

[0135] IκBα Ubiquitination Studies

[0136] In Vitro Assay:

[0137] IκBα and its phosphorylation situes mutants ³²S→³²A and ³⁶S→³⁶A(S32A-IκBα and S36A-IκBα) in pBluscript plasmids were generouslyprovided by T. Maniatis (Harvard University). Plasmid for theoverproduction of GST-Ub (pGES-2TK-Ub) was a gift of J. Huibregtse.GST-Ub was prepared as described in Scheffner et al., Cell 75: 495-505(1993).

[0138] Preparation of HeLa Cell Extract:

[0139] HeLa cells were harvested by centrifugation at 4,000 rpm. Thepacked cell volume was estimated, and the cells osmotically lysed by theaddition of 5 volumes of hypotonic buffer (10 mM Tris-Cl, pH 7.9, 1.5mMMgCl₂, 10 mM KCl, 0.2 mM PMSF, 0.5 mM DTT). The mixture was incubated onice for 10 minutes, then homogenized with 12 strokes of a Douncehomogenizer (B Type pestle), The crude lysate was centrifuged for 15minutes at 4,000 rpm, followed by recentrifugation of the supernatant at100,000×g for 30 minutes. This supernatant was concentrated by theaddition of solid (NH₄)₂SO₄ to 80% saturation, stirred at 4° C. for 30minutes and centrifuged for 15 minutes at 12,000 rpm. The precipitatewas resuspended in 1/5 volume of 20 mM Tris-Cl pH7.6, 20 mM KCl, 5 mMMgCl 2, 1 mM DTT, 0.5 mM PMSF, 5 μM Chymostatin and 20 μM E64, anddialyzed against >500 volumes of 20 mM Tris-Cl pH 7.6, 20 mM KCl, 5 mMMgCl2, 1 mM DTT, 10% glycerol at 4° C. overnight and stored at −70° C.until use.

[0140] Ubiquitination Assay:

[0141]³⁵S-IκBα and ³⁵S-S32A/S36 IκBα were prepared by coupled in vitrotranscription/translation in wheat germ extract (Promega, Madison, Wis.)using Trans³⁵S-Label (ICN Radiochemicals, Costa Mesa, Calif.) accordingto manufacturer's instructions. ³⁵S-IκBα and ³⁵S-S32A/S36A IκBα wereremoved from unincorporated radioactivity by gel filtration using aNICK-Spin column (Pharmacia Biotech, Piscataway, N.J.). ³⁵S-IκBα or³⁵S-S32A/S36A IκBα (˜40,000 cpm) were added to a 20 μl reactioncontaining 2 mM ATP, 10 mM creatine phosphate, 0.2 mg/ml creatinekinase, 70 μM GST-ubiquitin, 3.3 μM okadaic acid (Sigma, St. Louis,Mo.), 30 μm MG132, 2 μM ubiquitin aldehyde, 200 μM bestatin, 10 uM E64,0.5 mM PMSF, and 80 μg HeLa cell extract in 20 mM Tris-Cl pH 7.6, 20 mMKCl, 10 mM MgCl₂, 1 mM DTT, 10% glycerol. PR-39 or control peptides werealso added to individual reactions. The samples were incubated at 37° C.for 90 minutes followed by the addition Laemelli sample buffer to stopthe conjugation reactions. The samples were then heated for 5 minutes at95° C. and analyzed by SDA-PAGE on 4-15% acrylamide gradient gels(BioRad, Hurcules, Calif.). After electrophoresis, the gels wereincubated with gentle stirring in 30% methanol 10% acetic acid, driedand analyzed using a Fuji phosphorimager.

[0142] Cell Culture Assay:

[0143] ECV304 cells were stably transfected with a HA-tagged ubiquitinplasmid MT123 (kind gift of Dr. G. Walz, BIDMC). The clone with thehighest expression of HA-ubiquitin was treated with 10 μM PR39 for 45min followed by treatment with 1 ng/ml TNFα for 20 min. At that point,the cells were washed and lysed and IκBα was immunoprecipitated.Following SDS-PAGE and membrane transfer of the immunoprecipitatedmaterial, the HA-ubiquitinated IκBα was visualized by Western blottingwith an anti-HA antibody (Santa Cruz).

[0144] Electrophoretic Mobility Shift Assay

[0145] Nuclear extracts were prepared as described in Dyer, R. B. and N.K. Herzog, Biotechniques 19: 192-195 (1995). In brief, pancreatic tissuewas homogenized in ice cold 0.3M sucrose, cells were subjected toiso-osmolar lysis in buffer containing NP-40 and the isolated nuclei.Nuclear protein were extracted from intact nuclei in a buffer containing0.4 M KCl and stored in −70° C. before analysis.

[0146] Aliquots of 7.5-10 μg of nuclear protein wer mixed in 25 μlreactions containing 5 mM Tris pH 7.5, 100 mM NaCl, 1 mM DDT, 1 mM EDTA,4% (v/v) glycerol, 0.08 mg/ml salmon sperm DNA and H₂O. Theoligonucleotide probe (5′-AGT TGA GGG GAC TTT CCC AGG C-3′, Promega,Madison, Wis.) containing the kB binding motif was end labeled with[γ-³²P] ATP using T₄ polynucleotide kinase. 1×10⁶ cpm of the probe wasadded to the mixture, and the binding reaction was allowed to proceedfor 20 min at room temperature. The unlabeled oligonucleotide was usedin the specific competition assay. DNA-protein complexes were resolvedin a 6% non-denaturating polyacrylamide gel in a TBE buffer at 150V.Gels were dried and exposed to Kodak Bio Max MR film at −70° C.

Experiment 1

[0147] To identify possible intracellular targets of PR-39 peptideaction, a yeast two-hybrid screen of mouse cDNA library was performedusing the unique 4th exon DNA sequence of porcine PR-39 gene as “bait”.Four clones growing on selective media and demonstrating lacZ stainingwere purified and sequenced. All four clones encoded overlappingidentical cDNA sequences which are highly homologous to the sequence ofthe human α7 (HC8) subunit of the 20S proteasome (GeneBank accessionAF055983). The results are shown by FIG. 1 and reveals aPR-39/proteasome interaction and the consequential effect on IκBαdegradation and NFκB activity.

[0148]FIG. 1A shows the binding of PR-39 to α7 and its fragments in theyeast two-hybrid assay. Deletion mutants of the mouse α7 subunit werecloned into yeast two-hybrid vector, and the extent of growth of lacZ+colonies on selective medium following co-transformation with PR-39construct in the yeast CG1945 was determined. Note that only the fulllength a7 construct was able to bind to PR-39. Deletion analysis showedthat the presence of both the C-terminal as well as the N-terminalsequences of α7 are required for PR-39 peptide binding.

[0149] In order to confirm that PR-39 and the a7 subunit interact incells, a polyclonal anti-PR-39 antibody was used to immunoprecipitatePR-39 protein from EVC304 cells transfected with cDNA constructscorresponding either to the full length (EVC-PR-39) or exon 4 (39 aminoacid C-terminal domain) (ECV-E4) of the porcine PR-39 gene.

[0150]FIG. 1B shows the co-immunoprecipitation of PR-39 and 20S subunitsin ECV cells. Full-length porcine cDNA (containing the leader sequence)and a cDNA construct corresponding to the 4th exon of porcine PR-39 genewere cloned into eukaryotic expression vector pGRE5-2 (USB) and used tostably transfect an immortalized human endothelial cell line (ECV304,ATCC). For co-immunoprecipitation, wild type ECV, full-length PR-39(ECV-PR-39) and exon 4 PR-39 (ECV-E4) transfected cells were cultured in10%FBS-M199. Cells were lysed with the RIPA buffer, and equal amounts oftotal protein were pre-cleared with non-immune rabbit serum and proteinG plus/protein A-agarose beads (Calbiochem). The cleared samples wereincubated at 4° C. overnight with 20 μl of polyclonal anti-PR-39antibody and 40 μl of protein G plus/protein A-agarose beads or with thebeads alone. The beads were then washed three times with PBS,resuspended in Laemmli sample buffer (2% SDS, 10% glycerol, 0.5%β-mercaptoethanol, 0.004% bromphenol blue, 50 mM Tris-HCl pH 6.8),resolved on 10% SDA-PAGE and transferred to PVDF membrane and blottedwith 1:1000 mouse monoclonal anti-α7 or anti-α2 antibodies (AffinitiResearch Products Ltd., UK). As a control, the total cell lysate of ECVcells was subjected to SDS-PAGE and immunoblotting with anti-α7 mAb(last lane).

[0151]FIG. 1B also shows the presence of the α7 and α2 subunits in theimmunoprecipitate from ECV-PR-39 and ECV-E4 but not ECV cells as well asthe presence of additional bands likely corresponding to other 20Sproteasome subunits. Western blot analysis of the materialimmunoprecipitate from the whole cell lysate with the anti-α7 subunitmonoclonal antibody demonstrated the presence of a 29 kD bandcorresponding to the known size of the α7 proteasome subunit in theECV-PR-39 and ECV-E4, but not wild type ECV-304 cells.

[0152] In addition, evidence was obtained for the potential presence ofother 20S proteasome subunits (FIG. 1B). Western blotting with amonoclonal antibody directed against the α2 subunit of the 20Sproteasome demonstrated its presence in the anti-PR-39 antibodyimmunoprecipitate from the PR-39-expressing but not wild type ECV304cells (FIG. 1B). Taken together, these results demonstrate that PR-39can bind to the 20S proteasome particle via an interaction with the a7subunit.

[0153] Since such an interaction with a proteasome subunit may result ininhibition of proteasome-mediated protein degradation, the effect ofPR-39 expression on the degradation of IκBα, an important regulator ofgene transcription, was assessed. Stable expression of PR-39 constructsin ECV304 cells resulted in increased levels of IκBα.

[0154]FIG. 1C shows the stable expression of PR-39 increases IκBαexpression in ECV304 cells. For IκBα expression studies, normallyproliferating wild type ECV304 cells (ECV) or ECV-PR-39 and ECV-E4 cellswere lysed in the loading buffer, subjected to electrophoresis on 10%SDS-PAGE gel and transferred to a PVDF membrane. IκBα expression wasthen determined by Western blotting with anti-IκBα antibody (Santa Cruz,Inc.). ECV304 cells treated for 2 hr with 10 μM of lactacystin (LC) wereused as control. Note increased IκBα levels in ECV-E4 and ECV-PR-39cells as well as in lactacystin-treated cells.

[0155]FIG. 1D shows the dose-dependent effect of PR-39 treatment on IκBαprotein levels. Western blotting of ECV cell lysate was carried outfollowing 2 hr exposure to buffer (0) or 100 or 50 nM of PR-39. Note thedose-dependent increase in IκBα protein levels. Clearly, exposure ofwild type ECV304 cells to the synthetic PR-39 peptide led to adose-dependent increase in IκBα levels.

[0156] Tumor necrosis factor (TNF)-α is known to induce rapiddegradation of IκBα by the Ub-proteasome pathway. To test whether PR-39can inhibit TNF-α induced degradation of IκBα, a human monocytic cellline U937 was employed that normally exhibits significant baselinelevels of IκBα.

[0157] As shown by FIG. 1E, PR-39 prevents TNF-a induced degradation ofIκBα. U937 cells were cultured in 10% FBs-RPMI medium in the absence (−)or presence of TNF-a (1 ng/ml). 10 min following TNF-α addition, thecells were washed and IκBα protein levels assessed by Western blotting.

[0158] The results show TNF-α caused a rapid decline of the level ofIκBα in these cells, and this effect was markedly reduced bypretreatment with PR-39 or proteasome inhibitos lactacystin or MG132(FIG. 1E). Note that a complete disappearance of IκBα occurs in TNF-αtreated cells with 45 min of pretreatment with 100 nM of PR-39 or 10 μMof MG132 or lactacystin (LC) prior to addition of blocked TNF-α, blockedcytokine-induced degradation of IκBα expression.

[0159] To test whether treatment with PR-39 peptide irreversibly blockedIκBα degradation, U937 cells were pretreated with the peptide and thenwere extensively washed, and 45 min later exposed to TNF-α. In parallelcells were exposed to MG132, a rapidly reversible competitive inhibitorof the proteasome or with lactacystin which covalently and irreversiblymodifies the active site threonine residues. The results are shown byFIG. 1F.

[0160]FIG. 1F demonstrates the reversibility of PR-39 inhibition of IκBαdegradation. U937 cells were pretreated with 100 nM of PR-39, 10 μM ofeither MG132 or lactacystin (LC) or buffer (Control) for 45 min. Thesecells were then extensively washed with fresh medium. 45 min later TNF-α(1 ng/ml) was added to medium, and the extent of IκBα degradation wasdetermined 10 min later by Western blotting. Such Western blot analysisdemonstrated the complete disappearance of IκBα protein in PR-39 and MG132-treated cells while pretreatment with lactacystin irreversiblyblocked proteasome-mediated degradation of IκBα.

Experiment 2

[0161] To show that the PR-39 peptide not only prevented inhibition ofIκBα degradation but also inhibited NFκB-dependent transcription in cellculture and in mice (i.e., that the IκBα protein was functional), ECV304cells were transiently transfected with a reporter construct containinga tandem of four NFκB binding sites in front of a minimal TK promoterdriving luciferase cDNA. The results are presented by FIG. 2A.

[0162]FIG. 2A shows that PR-39 administration blocks NFκB-dependent geneexpression in cell culture. The effect of PR-39-mediated inhibition ofIκBα degradation on NFκB-dependent transcription was tested in ECV304cells transiently transfected with pNFκB-Luc reporter vector. Exposureto 1 ng/ml TNF-α led to a significant increase in luciferase activitythat was completely blocked by 45 min of pretreatment with 10 nM ofPR-39 or 10 μM of either MG132 (MG) or lactacystin (LC)*p<0.01 vs.control (luciferase activity in the absence of TNF-α).

[0163] To study whether PR-39 can also inhibit NFκB activation in intactanimals, two mouse models of acute injury were used—acute pancreatitisand acute myocardial infarction. The results are shown by FIGS. 2B and2C respectively.

[0164]FIG. 2B demonstrates that PR-39 blocks IκBα degradation in mousepancreas following induction of acute pancreatitis. Male mice weighing20-25 g (ICR, Charles River Laboratories, Wilmington, Mass.) werepretreated with PR-39 (10 mg/kg, i.v.) or physiological saline 1 hourbefore injecting 50 μg/kg i.v. of caerulein (Research Plus, Bayonne,N.J.) to induce acute pancreatitis. 30 min later the animals weresacrificed in a CO₂ chamber and a 75-100 mg piece of pancreas wasremoved for Western blotting and NFκB gel shift assays. Notepreservation of IκBα in both PR-39-treated animals.

[0165]FIG. 8C shows NFκB-dependent transcription in the mouse pancreas.NFκB activation in mouse pancreatic tissues in the setting ofcaerulein-induced pancreatitis was examined using electromobility shiftassay. Note almost complete disappearance of NFκB gel shift in PR-39treated animals. 100×excess of oligonucleotide competitor of NFκBbinding site was used to demonstrate band specificity (κB oligo).

[0166] As empirically shown, induction of acute pancreatitis bytreatment with the cholecystokinin analog caerulein leads to the promptdisappearance of IκBα (FIG. 2B) and activation of NFκB-dependenttranscription in the pancreas (FIG. 8C). Intravenous injection of 10mg/kg of PR-39 one hour prior to caerulein administration largelyprevented IκBα degradation and, consequently, NFκB-dependent geneexpression as assessed by DNA gel shifts.

[0167] NFκB-dependent transcription is also activated followinginduction of ischemia in the heart, leading to increased expression ofseveral adhesion molecules, including ICAM-1 and VCAM-1. To test theeffect of PR-39 peptide on NFκB-dependent gene expression in this model,we assessed VCAM-1 and ICAM-1 protein levels following induction ofmyocardial infarction in transgenic mice expressing PR-39 cDNA incardiac myocytes (αMHC-PR-39) and age-matched control mice. The resultis given by FIG. 2D.

[0168]FIG. 2D shows that PR-39 blocks NFκB-dependent gene expression ina mouse infarct model. Transgenic mice stably expressing PR-39 incardiac myocytes (αMHC-PR-39 mice) or litter mate controls (controlmice) were subjected to acute coronary artery ligation as described(30)and randomized to intraperitoneal implantation of Alzet minipumps (AlzaCorp, Palo Alto, Calif.) delivering 1 μg/kg/24 hr of PR-39 or buffer.Myocardial tissues collected 1, 3 or 7 days later were subjected toWestern blot analysis for ICAM-1 and VCAM-1 gene expression that werenormalized to expression levels prior to induction of infarction. Notethe rapid increase in both ICAM-1 and VCAM-1 expression in control butnot αMHC-PR-39 mice.

[0169] The levels of both proteins increased significantly followingmyocardial infarction in control mice by 24 hr, reaching a peak by 72 hrand then gradually declined. In contrast, induction of myocardialinfarction had no effect on either ICAA-1 or VCAM-1 protein levels inαMHC-PR-39 mice.

Experiment 3

[0170] One possible explanation of the PR-39-dependent inhibition ofIκBα degradation would be that the peptide like MG132 reversiblyinhibits all proteasome function, thus generally suppressingintracellular protein degradation. Therefore the extent of degradationof long-lived cell proteins before or following exposure was compared toeither PR-39 or MG132. The result is given by FIG. 3A.

[0171]FIG. 3A shows that PR-39 does not substantially affectproteasome-dependent degradation of total cellular proteins.Exponentially proliferating U937 cells were grown in 10% FBS-RPMI 1640methionine-free medium supplemented with 200 μCi ³⁵S Met for 16 hr.Cells were then washed with complete RPMI 1640 medium 3 times andcultured in 10% FBS-RPMI (chase) in the presence of 10 μM of eitherPR-39 or MG132 or an equal volume of PBS. Chloroquin (45 μM) was addedto all cultures to prevent the lysosomal protein degradation. 1 hr laterthe cells were lysed, and TCA soluble ³⁵S counts were measured in aliquid scintillation counter (LKB). All experiments were carried out intriplicate and repeated three times. The data are shown as mean±standarddeviation.

[0172] Note significantly lower inhibition of total cell proteindegradation by PR-39 than by MG132 in FIG. 3A. After 16 hr metaboliclabeling of total cell protein in ECV304 cells, MG132 treatment resultedin approximately 50% inhibition of total cell protein degradation, inaccordance with previously reported results. In contrast, administrationof PR-39 had little or no effect on this process.

[0173] To confirm that PR-39 does not cause a general inhibition ofprotein degradation, the expression level of HSP70, a major heat shockprotein whose expression is stimulated by proteasome inhibitors, wasstudied. FIG. 3B shows the lack of induction of HSP70 expression inPR-39-treated cells. Western blotting of cell extract from untreated(control) and PR-39-(1μM) or MG132-(1 μM) treated U937 cellsdemonstrated the appearance of HSP70 expression after 3 hr exposure toMG132 but not PR-39. Western blot analysis demonstrated a strikingincrease in HSP70 levels in cultured ECV304 cells following treatmentwith 10 μM of MG132. However, exposure to a similar concentration ofPR-39 failed to stimulate HSP70 accumulation.

[0174] In addition, studies in ECV304 cells with specific antibodiesfailed to demonstrate any effect of PR-39 treatment on the levels ofseveral proteins principally controlled by proteasome-dependentdegradation including the cell cycle inhibitor p21 and cyclin E andtranscription factor c-fos. This is shown by FIG. 3C.

[0175] Note that FIG. 3C shows the effect of PR-39 administration onproteasome-dependent protein levels in U937 cells. The level ofproteasome-regulated cell cycle repressor p21, cyclin E and c-fosproteins were determined by Western blotting in U937 cells pretreatedwith various amounts of PR-39 and then exposed to TNF-α (1 ng/ml). Thelack of PR-39 dependent inhibition of proteasome-mediated degradation ofthese proteins is demonstrated.

[0176] Prolonged exposure to proteasome inhibitors tend to induceapoptosis in cell culture. To assess the potential cytotoxicity ofPR-39, ECV304 cells were cultured in the presence of 10 μM of PR-39,MG132 or lactacystin.

[0177]FIG. 3D shows that PR-39 does not affect cell growth in culture.50,000 ECV304 cells were plated in 6 well plates in 10% FBS-M199 andsynchronized by serum deprivation in 0.5% FBS-DMEM for 48 hr. At the endof that time, cell culture medium was changed to 10% FBS-M199 and 10 μMof PR-39, lactacystin or buffer (control) were added. Forty eight hourslater cell counts were determined using a Coulter Counter (CoulterInstruments, Inc.). All experiments were performed in triplicate andrepeated 2 times (mean±SD). Note the markedly decreased cell count inlactacystin-treated culture and the lack of significant growthinhibition by PR-39 revealed by FIG. 3D.

[0178] Moreover, the cell counts (48 hrs) documented that, whileaddition of PR-39 did not affect the cells' ability to proliferate,exposure of these cells to either MG132 or lactacystin led to asignificant decline in cell counts. These results demonstrate that PR-39peptide is able to inhibit IκBα degradation without significantlyaffecting overall protein degradation in cells.

Experiment 4

[0179] The preceding experiments suggest that PR-39 peptide inhibitsIκBα degradation in a selective manner. To explore potential mechanismsof this selective inhibition, the effect of peptide administration onIκBα phosphorylation and ubiquitination was studied. It is known thatTNF-α treatment leads to phosphorylation of IκBα, which is required forits subsequent ubiquitination. The result is indicated by FIG. 4A.

[0180]FIG. 4A shows the effect of PR-39 on IκBα phosphorylation in cellculture. U937 cells cultured in the absence (−) or presence (+) ofpretreatment with 10 μM PR-39 were exposed to 1 ng/ml TNFα. 45 min laterthe cells were lysed and subjected to SDS-PAGE and Western blotting withanti-IκBα antibody. Note the increased amount of the typical-appearingphosphorylated IκBα band in PR-39 treated cells.

[0181] Western blotting of U937 cell lysates after in vivo ³²p labelingdemonstrated the appearance of the phosphorylated form of IκBα followingTNF-α exposure. Pretreatment with PR-39 increased the amount of thephosphorylated form of IκBα presumably due to inhibition of itsdegradation.

[0182] To test the effect of PR-39 on IκBα ubiquitination in a cell freesystem, HeLa cell extract was used to ubiquitinate in vitro transcribed,translated and phosphorylated IκBa. FIG. 4B shows the effect of PR-39 onIκBα ubiquitination in vitro. In vitro ubiquitination of phosphorylatedIκBα protein was carried out using crude HeLa cell extract in thepresence of a control peptides (lanes 1 and 2), PR-39 peptide (lanes 3and 4) or in the absence of both (lane 5). Note that the addition ofeither PR-39 peptide at two different concentrations or of a control(random) peptide failed to affect IκBα ubiquitination. At the same time,no ubiquitination of phosphorylation site IκBα mutants (lanes 6 and 7)or unphosphorylated IκBα (wt IκBα, lane 8) was detected.

[0183] Clearly, therefore, in the absence of PR-39 incubation ofphosphorylated IκBα protein with a HeLa extract led to the appearance ofmultiple high molecular weight uniquitinated forms of IκBα. The additionof PR-39 peptide in an amount sufficient to inhibit IκBα degradation incell culture had no effect on the extent of IκBα ubiquitination in thisassay.

[0184] To assess the effect of PR-39 peptide treatment on IκBαubiquitination in intact cells, a stable ECV304-derived cell lineexpressing HA-tagged ubiquitin was generated. FIG. 4C shows the effectof PR-39 on IκBα ubiquitination in cell culture. HA-Ub expressing ECV304cells were exposed to 1 ng/ml TNF-α in the presence (+) or absence (−)of 10 μM PR-39 pretreatment. 45 min later the cells were lysed andsubjected to IκBα immunoprecipitation followed by Western blotting ofthe pellet material with anti-HA antibody. Note the presence of multipleubiquitinated IκBα intermediaries in PR-39 peptide treated cells but notin control cells.

[0185] Immunoprecipitation of IκBα followed by Western blotting withanti-HA antibody thus demonstrated the accumulation of moreubiquitinated IκBα complexes in cells pretreated with 10 μM of PR-39than in untreated cells. These findings together argue that PR-39 doesnot block the ubiquitination process but does inhibit the degradation ofubiquitinated IκBα by the 26S proteasome.

[0186] A recent study [Dai et al., J. Biol. Chem. 273: 3562-3573 (1998)]suggested that binding of the ubiquitinated IκBα to VCP, a 26Sproteasome associated protein, is necessary but not sufficient forsubsequent IκBα degradation. FIG. 4D shows that PR-39 does not inhibitIκBα-VCP binding. Wild type ECV304 cells (treated as described in FIG.4C) were subjected to immunoprecipitation with anti-IκBα antibody(IκBα-IP) followed by Western blotting with anti-VCP-3 antibody (1:3000,dilution; a kind gift of Dr. C. C. Li (NIH)). Note the presence of 90 kDVCP band in the presence or absence of PR-39 treatment.

[0187] The results of FIG. 4D reveal that Western blotting with theanti-VCP antibody demonstrated the presence of this protein in theanti-PR-39 antibody immunoprecipitate of the ECV-PR-39 cells (notshown). Western blot analysis of anti-IκBα antibody immunoprecipitatedmaterial from ECV304 cells demonstrated the presence of VCP in accordwith the previously reported study. Pretreatment with PR-39 increasedthe amount of VCP in the IκBα immunoprecipitate (FIG. 4D) as would beexpected given the increased amount of ubiquitinated forms of IκBα inthe PR-39-treated cells. Accordingly, PR-39 does not interfere withIκBα-VCP binding.

Conclusions Drawn from Experimental Series

[0188] 1. The empirical results presented several types of evidence thatthe naturally occurring anti-bacterial 39 amino acid peptide, PR-39,selectively inhibits IκBα degradation in cultured cells and in twodifferent mice models. Stable expression of this PR-39 peptide resultedin increased IκBα content in cell culture; while pretreatment ofcultured cells with the PR-39 peptide inhibited TNF-a-induceddegradation of IκBα and abolished induction of NFκB-dependenttranscriptional activity. Moreover, pretreatment with the PR-39 peptideblocked IκBα degradation and activation of NFκB-dependent transcriptionin the mouse pancreas following purposeful induction of chemicalpancreatitis. Finally, transgenic expression of PR-39 peptide in mousecardiac myocytes prevented expression of NFκB-dependent endothelialadhesion genes VCAM-1 and ICAM-1 in the clinical setting of acutemyocardial ischemia.

[0189] 2. While the ability to inhibit IκBα degradation in-situ mayimply that PR-39 peptide acts as a proteasome inhibitor, severalempirical observations clearly set it apart from all the small moleculeproteasome inhibitors described to date. Most important in this regardis the selective nature of the PR-39 peptide effect. This selectivity isindicated: (a) by the lack of any effect on overall proteasome-dependentprotein degradation in cells; (b) by the absence of induction of heatshock proteins such as HSP70; and (c) by the lack of toxicity followinga long-term exposure in cell culture or transgenic expression in mice.In contrast, exposure of cultured cells to the proteasome inhibitors,MG132 and lactacystin, leads to a general reduction in intracellularproteolysis, a rapid increase in HSP70 expression and cell death (asreported in the scientific literature). Furthermore, known directinhibitors of 20S proteasome affect its active sites and therefore theinhibition of proteolysis as an event is not specific to any particularprotein or class of proteins.

[0190] 3. Selectivity as a characteristic effect can be achieved,however, as an indirect action by blocking substrate-specific steps inthe degradative pathway. Experiments were performed to determine whetherthe selective inhibition by PR-39 peptide of IκBα degradation is due toinhibition of known pathway steps required for its degradation,including phosphorylation, ubiquitination and VCP binding. Studies inthe cell free-system failed to show any effect of PR-39 peptidetreatment on ubiquitination of IκBα, while the cell culture experimentsdemonstrated an accumulation of phosphorylated and ubiquitinated formsof IκBα in the PR-39 peptide treated cells. Furthermore, PR-39 peptidedid not interfere with the binding of ubiquitinated IκBα to VCP. Thesefindings evidence an interaction mechanism in which PR-39, whileattached to the 26S proteasome, prevents recognition or binding of theubiquitinated IκBα-VCP complex or blocks this complex's translocationinto the 26S proteasome.

[0191] 4. Several contrasting experimental results described by theExperimental Series herein are also consistent with such an action ofPR-39 on the 26S proteasome. The PR-39 peptide is able to bind with highaffinity directly to the α7 subunit of the proteasome, as shown in theyeast two-hybrid assay as well as by co-immunoprecipitation with theanti-PR-39 antibody. Furthermore, as a result of such direct binding toα7, PR-39 peptide is able to bring down at least one other (α2) 20Ssubunit (and probably other subunits as well) indicating that thepeptide binds to the whole 20S particle without causing itsdissociation. While the precise site of interaction on the α7 subunit oron PR-39 has not yet been fully established, the high negative charge ofthe C-terminal end of the α7 subunit which makes it a likely site forbinding of the postively charged PR-39 molecule. The α7 C-terminalsequence is actually the most negatively charged sequence of any of theproteasome a subunits; and it has the ability to assemble intoheptameric ring structures by itself as well as to induce ring formationof other a subunits.

[0192] 5. However, it is unclear as yet precisely how PR-39 interactionwith the 26S proteasome causes the observed selective inhibition of thedegradation of phosphorylated and ubiquitinated IκBα. One mode ofpossible mechanism would interfere with the binding of the ubiquitinatedIκBα-VCP complex to the 26S proteasome. The observed inhibition of theNFκB-dependent transcription in PR-39 peptide treated cells or animalsis consistent with this mechanism of action since inhibition of VCP-IκBαbinding to the proteasome would leave the ubiquitinated IκBα in complexwith NFκB. Alternatively, PR-39 peptide may interfere with another, yetunidentified, pathway step involved in IκBα degradation by theproteasome. Another possibility is that PR-39 binding to the α7 subunitmay alter the 3-dimensional proteasome architecture or interaction ofthe 20S particle and the 19S regulatory subunit that would affect theentry of certain protein substrates into the 20S cylinder. All of thesepossible modes of action are consistent with the empirical observationthat PR-39 inhibition of IκBα degradation is dose-dependent andreversible, apparently requiring the continuous presence of the PR-39peptide.

[0193] The present invention is not to be limited in form nor restrictedin scope except by the claims appended hereto.

What we claim is:
 1. A method for selectively inhibiting degradation ofIκBα within a targeted collection of viable cells in-situ, said methodcomprising the steps of: identifying a collection of cells comprisingviable cells in-situ as a target for inhibiting IκBα degradation;providing means for effecting an introduction of at least one memberselected from the group consisting of the PR-39 oligopeptide collectiveto the cytoplasm of said targeted collection of cells; introducing atleast one member of the PR-39 oligopeptide collective to the cytoplasmof said targeted collection of cells using said effecting means;allowing said introduced PR-39 oligopeptide collective member tointeract with such IκBα and proteasomes as are present within thecytoplasm of said targeted collection of cells whereby (a) at least someof the proteasomes interact with said PR-39 oligopeptide collectivemember, (b) at least a part of the proteolytic activity mediated by saidproteasomes becomes selectively altered by said interaction, and (c) theselectively altered proteolytic activity of said proteasomes results ina marked inhibition of IκBα degradation in-situ within the cytoplasm ofsaid targeted collection of viable cells.
 2. A method for decreasing theactivity of NFκB transcription factor within a targeted collection ofviable cells in-situ, said method comprising the steps of: identifying acollection of cells comprising viable cells in-situ as a target fordecreased NFκB activity; providing means for effecting an introductionof at least one member selected from the group consisting of the PR-39oligopeptide collective to the cytoplasm of said targeted collection ofcells; introducing at least one member of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells usingsaid effecting means; allowing said introduced PR-39 oligopeptidecollective member to interact with such IκBα and proteasomes as arepresent within the cytoplasm of said targeted collection of cellswhereby (a) at least some of the proteasomes interact with said PR-39oligopeptide collective member, (b) at least a part of the proteolyticactivity mediated by said proteasomes becomes selectively altered bysaid interaction, (c) the selectively altered proteolytic activity ofsaid proteasomes results in a marked inhibition of IκBα degradationin-situ within the cytoplasm of said targeted collection of viablecells, and (d) said reduction of IκBα degradation results in a decreasein activity for such NFκB transcription factor as is presentintracellularly.
 3. A method for selective control of NFκB-dependentgene expression in-situ within a collection of viable cells, said methodcomprising the steps of: identifying a collection of cells comprisingviable cells in-situ as a target for controlling NFκB-dependent geneexpression; providing means for effecting an introduction of at leastone member selected from the group consisting of the PR-39 oligopeptidecollective to the cytoplasm of said targeted collection of cells;introducing at least one member of the PR-39 oligopeptide collective tothe cytoplasm of said targeted collection of cells using said effectingmeans; allowing said introduced PR-39 oligopeptide collective member tointeract with such IκBα and proteasomes as are present within thecytoplasm of said targeted collection of cells whereby (a) at least someof the proteasomes interact with the PR-39 oligopeptide collectivemember, (b) at least a part of the proteolytic activity mediated by saidproteasomes becomes selectively altered by said interaction, (c) theselectively altered proteolytic activity of said proteasomes results ina marked reduction of IκBα degradation in-situ within the cytoplasm ofsaid targeted collection of cells, (d) said reduction of IκBαdegradation results in a decrease in activity for such NFκBtranscription factor as is present intracellularly, and (e)NFκB-dependent gene expression is selectively controlled in saidtargeted collection of viable cells.
 4. The method as recited in claim1, 2 or 3 wherein said collection of viable cells includes at least onetype of cell selected from the group consisting of endothelial cells,myocytes and myoblasts, fibrocytes and fibroblasts, epithelial cells,osteocytes and osteoblasts, neuronal cells and glial cells,erythrocuctes, leukocytes, and progenitor cells of all types.
 5. Themethod as recited in claim 1, 2 or 3 wherein said collection of cellscomprises at least one tissue selected from the group consisting ofmyocardium, skeletal muscle, smooth muscle, an artery, a vein, lung,brain, kidney, spleen, liver, gastrointestinal tissue, nerve tissue,limbs, and extremities.
 6. The method as recited in claim 1, 2 or 3wherein the means for an introduction of a PR-39 oligopeptide collectivemember include one selected from the group consisting of catheter-basedintroduction means, injection-based introduction means, infusion-basedintroduction means, localized intravascular introduction means,liposome-based introduction means, receptor-specific peptideintroduction means, slow releasing means for peptide secretion in livingcells and sequested organisms.
 7. The method as recited in claim 1, 2 or3 wherein the means for an introduction of a PR-39 oligopeptidecollective member includes the DNA sequences coding for PR-39oligopeptides of different sizes inserted in a suitable vector fortransfection and subsequent expression of peptides within said cells. 8.The method as recited in claim 1, 2 or 3 wherein said method ispracticed under in-vivo conditions.
 9. The method as recited in claim 1,2 or 3 wherein said method is practiced under in-vitro conditions.
 10. Afamily of PR-39 derived oligopeptides whose members individually cause aselective inhibition of proteasome-mediated IκBα degradation in-situafter introduction intracellularly to a viable cell, each member of saidoligopeptide family being: a peptide less than 39 amino acid residues inlength; at least partially homologous with the N-terminal amino acidresidue sequence of the native PR-39 peptide; able to interact in-situwith such IκBα as is present within the cytoplasm of the cell; and ableto inhibit markedly the degradation activity of proteasomesintracellularly as a consequence of said interaction with IκBα.
 11. ThePR-39 derived oligopeptide family as recited in claim 10 whosemembership includes a peptide comprised of 15 amino acid residues whosesequence is Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg-Pro-Arg-Pro-Pro.12. The PR-39 derived oligopeptide family as recited in claim 10 whosemembership includes a peptide comprised of 11 amino acid residues whosesequence is Arg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr-Leu-Pro-Arg.
 13. The PR-39derived oligopeptide family as recited in claim 10 whose membershipincludes a peptide comprised of 8 amino acid residues whose sequence isArg-Arg-Arg-Pro-Arg-Pro-Pro-Tyr.